We have analyzed the transcriptional efficiencies of one downstream and two upstream early simian virus 40 (SV40) transcripts synthesized in vitro. Our studies have demonstrated that the presence of upstream AUG initiation codons decrease the initiation of translation at the early SV40 gene products initiation. Labeling of in vitro translation products with proline and serine in an in vitro translation reaction programmed with capped SV40 RNAs resulted in a synthesis of a small protein of 2.7-Kd. In vivo labeling of SV40 infected CV-1 cells demonstrated the accumulation of a peptide of similar size at late times after infection. The function of this novel protein in the lytic cycle of SV40 is currently under investigation. We have employed mutants in the region encoding the carboxy terminus of T-antigen to further investigate a role of T-antigen in SV40 late gene expression. While these mutants have little effect on the efficiency of viral DNA replication, they significantly decrease the yield of infectious virus particles by 3-4 logs. The level of late viral RNA and capsid protein (VP1, VP2 and VP3) are 5- to 15-fold lower in these mutants in comparison to those in the wild-type. Analysis of another late viral polypeptide, the agnoprotein, has demonstrated a significant decrease (over 100-fold) in its synthesis of the 61-amino acid polypeptide.